Staphylococcus aureus is responsible for numerous difficult-to-treat infections. Antibiotic treatment failure is common and often not attributable to antibiotic resistance. Currently, there is an over-reliance on susceptibility assays involving rich media. These conditions are drastically different to those experienced by bacteria during infection. The identification of novel extrinsic factors that influence S. aureus antibiotic susceptibility in vivo will likely improve treatment outcomes. Our preliminary data show that a sub-population of S. aureus cells survive phagocytosis by macrophages, are coerced into a low-metabolic state by host- produced oxidative stress and these cells survive subsequent killing by antibiotics. We hypothesize that these cells represent an important niche for the dissemination of bacteria and the establishment of infection. However, determining whether a S. aureus cell has encountered oxidative stress over the course of infection is currently not possible. Analysis of particular transcripts or proteins only give a sense of the stresses experienced by the bacteria at a particular instant in time. We propose engineering a strain of S. aureus with a stress-activated memory element to detect if oxidative stress has ever been encountered by S. aureus cells through the development of infection. We propose integrating a two-part genetic circuit, termed FLIPPER, onto the chromosome of S. aureus. The tool consists of an oxidative stress-activated promoter upstream of a unidirectional recombinase; when expressed, the recombinase binds to two recognition sites and flips the intervening antibiotic resistant cassette into the ON orientation. The unidirectional recombinase elicits permanent genetic memory of stress encountered by surviving cells and their progeny. In this proposal, we aim to build and optimize FLIPPER for application in future projects. Flipping activity will be tested in vitro and tuned by modulating the translation rate of the recombinase using the tool Next Generation RBS calculator V2. The strain with the best flipping activity (strain that flips ON in response to stress but remains OFF in the absence of stress) will be used to infect macrophages. Flipping activity will be determined in macrophages with and without a functional oxidative burst and after treatment with antibiotics. Successful completion of this proposal will yield a tool that we can apply to answer two questions in downstream projects: 1) have S. aureus cells that successfully established infection encountered oxidative stress? and 2) have antibiotic tolerant persister cells in vivo encountered oxidative stress? The construction of FLIPPER will allow us to ask these questions in a unique and powerful way that, that is unachievable with existing technology. Furthermore, FLIPPER will be pliable for use by other researchers to answer a myriad of important questions.